Novel interferon-alpha

ABSTRACT

A novel human IFN-α subtype and its derivative having an unprecedentedly high specific activity, DNA encoding these proteins, an expression vector having said DNA, a transformant transformed with said expression vector, a method of producing the above human IFN-α and its derivative, and medical uses of the above human IFN-α and its derivative.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This is a divisional of application Ser. No. 09/889,035, filedJul. 11, 2001, which is a National Stage Application filed under § 371of PCT Application No. PCT/JP00/00015, filed Jan. 5, 2000; the abovenoted prior applications are all hereby incorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates to a novel interferon-α(hereinafter referred to as IFN-α). More preferably, the presentinvention relates to a novel human IFN-α and its derivative having anunprecedentedly high specific activity, and a gene thereof, as well asmedical uses of said IFN-α and its derivative.

BACKGROUND ART

[0003] IFN is a generic term for proteins having anti-viral activity,among which those produced from leukocytes or lymphoblastic cells bystimulation with virus or double stranded nucleic acids are termed asIFN-α. IFN-α has a variety of activities including anti-viral activityand a cellular growth-suppressing activity, which activities have beenfound to be useful in a variety of diseases such as hepatitis type B,hepatitis type C, and cancer.

[0004] Analysis of base sequences of IFN-α genes cloned from a varietyof DNA libraries have revealed that IFN-α has several subtypes (Science209: 1343-7 (1980), Gene 11: 181-6 (1980), Nature 290: 20-26 (1981),Nature 313: 698-700 (1985), J. Invest. Dermatol. 83: 128s-136s (1984)).For example, for the main subtype gene of IFN-α2, three types (α2a, α2b,and α2c) have been identified (J. Interferon Res. 2: 575-85 (1982), J.Interferon Res. 13: 227-31 (1993), J. Biol. Chem. 268: 12565-9 (1993),Acta Virol. 38: 101-4 (1994), Biochim. Biophys. Acta. 1264: 363-8(1995)). In addition, there are currently known nearly 20 types ofsubtype genes including IFN-α1a, -α1b, -α4a, α4b, -α5, -α6, etc.

[0005] On the other hand, vigorous efforts have been made in structuralanalysis of proteins in stead of genes, that is to purify each subtypeof natural IFN-α and then to analyze its primary structure. A group inWellcome, for example, made an attempt on structural analysis using amixture of two fractions separated by gel filtration of purified IFNderived from Namalwa cells, human lymphoblastic cells, and, as a result,have demonstrated the structure, though not complete, of IFN-α1 andIFN-α2 (Nature 287: 408-11 (1980)). As a result of intensive efforts topurify Namalwa cell-derived IFN subtypes, Zoon et al. of FDA havesuccessfully isolated several subtypes and revealed their partialstructure, anti-viral activity, cellular growth-suppressing activity,and NK cell-inducing activity (Infect. Immun. 34: 1068-70 (1981), J.Biol. Chem. 267: 15210-6 (1992), J. Biol. Chem. 268: 12591-5 (1993)).Furthermore, in the analysis of the primary structure for one majorsubtype, they have demonstrated that it was IFN-α2b (J. Biol. Chem. 267:15210-6 (1992)).

[0006] As stated above, IFN-α has various subtypes, of which basesequences and amino acid sequences are being elucidated, though thestructure and physical properties of all subtypes have not beenrevealed.

DISCLOSURE OF THE INVENTION

[0007] The present invention intends to provide a novel IFN-α and itsgene. Thus, the present invention intends to provide a novel humanIFN-α, its derivative having an unprecedentedly high specific activity,a gene encoding them, and a pharmaceutical agent comprising said IFN-αand its derivative as active ingredient.

[0008] The inventors of the present invention have attempted to isolatemajor subtypes contained in IFN-α derived from human natural-typelymphoblastic cells (hereinafter referred to as HLBI). Thus, theinventors have found that the subtypes can be easily separated by meansof a reverse-phase HPLC that utilizes μBondasphere column and Vydac-C4column and thereby have successfully isolated and purified 12 majorsubtypes contained in HLBI.

[0009] From the analysis of the N-terminal amino acid sequence and theprimary structure of the isolated subtypes, it was found that a novelIFN-α subtype was contained in addition to the existing IFN-α1, α2b, α5,α7, α8, α14, α17 and α21. The inventors of the present invention havetermed this novel IFN-α subtype as IIIe.

[0010] On these subtypes, anti-viral activity against Sindbis virus wasdetermined using human-derived cultured cells, FL cells, and it wasfound that the anti-viral activity of a major subtype IFN-α2b was1.67×10⁸ u/mg whereas the novel IFN-α subtype IIIe had the highest andunprecedentedly high specific activity of 4.3-5.2×10⁸ u/mg.

[0011] Furthermore, the identification of the entire amino acid sequenceof the subtype IIIe revealed that the primary structure of the subtypeIIIe was similar to an amino acid sequence deduced from the sequence ofIFN-α10a (=−αC) gene as reported in Nature 1981 Mar. 5; 290, 20-26, buthad a novel amino acid sequence in which the amino acid at position 19was Ala in stead of Gly. The cloning of said IIIe gene also revealedthat it is different from IFN-α10a by three bases on the base sequencelevel.

[0012] As described above, the IFN-α subtype IIIe of the presentinvention has an unprecedentedly high specific activity, and thereby itsdosage can possibly be reduced compared to commercially availablerecombinant human IFN-α2a, recombinant human IFN-α2b, etc. Furthermore,it is expected to exhibit effectiveness on cases with HCV-Genotype II,high virus level etc. on which conventional IFN is believed to be notvery effective.

[0013] The present invention was completed based on the above findings.

[0014] Thus, the present invention relates to the following (1) to (13):

[0015] (1) DNA comprising the base sequence as set forth in SEQ ID NO: 1or SEQ ID NO: 2, or DNA encoding a protein comprising the amino acidsequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4;

[0016] (2) DNA encoding a derivative of human interferon-α, said DNAbeing selected from

[0017] (A) DNA hybridizing to the DNA according to the above (1) under astringent condition, and

[0018] (B) DNA encoding a protein in which one or a plurality of aminoacid residues of a protein encoded by the DNA according to the above (1)have been replaced, deleted, and/or added,

[0019] wherein the protein encoded by said DNA has the followingcharacteristics (a) and (b):

[0020] (a) having a specific activity higher than 4.0×10⁸ units/mg asmeasured by an anti-viral activity assay on Sindbis virus using the FLcell, a human-derived cultured cell; and

[0021] (b) migrating as a band with an apparent molecular weight of 20kDa-23 kDa on a sodium dodecyl sulfate-polyacrylamide gelelectrophoresis after reduction treatment;

[0022] (3) The DNA according to the above (2) which encodes a proteincomprising an amino acid sequence in which 1-5 amino acid residues inthe amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4have been replaced, deleted, and/or added;

[0023] (4) An expression vector having the DNA according to any one ofthe above (1)-(3);

[0024] (5) A transformant transformed with the expression vectoraccording to the above (4);

[0025] (6) A method of producing a recombinant human interferon-α or itsderivative, which method comprises culturing the transformant accordingto the above (5) and recovering the expressed recombinant humaninterferon-α or its derivative;

[0026] (7) A human interferon-α or its derivative which is encoded bythe DNA according to any one of the above (1)-(3) or produced by theproduction method according to the above (6);

[0027] (8) A human interferon-a comprising the amino acid sequence asset forth in SEQ ID NO: 3 or SEQ ID NO: 4;

[0028] (9) A human interferon-α or its derivative according to the above(7) or (8) or a pharmaceutically acceptable salt thereof for use asactive ingredient of a pharmaceutical composition;

[0029] (10) A pharmaceutical composition comprising the humaninterferon-α or its derivative according to the above (7) or (8) or apharmaceutically acceptable salt thereof as active ingredient togetherwith a pharmaceutically acceptable carrier or excipient;

[0030] (11) The pharmaceutical composition according to the above (10)which is for treatment of viral diseases;

[0031] (12) The pharmaceutical composition according to the above (10)which is for treatment of cancer;

[0032] (13) A method of treating viral diseases or cancer which methodcomprises administering to a mammal including a human an effectiveamount of the human interferon-α or its derivative according to theabove (7) or (8) or a pharmaceutically acceptable salt thereof.

BEST MODE FOR CARRYING OUT THE INVENTION

[0033] The DNA of the present invention encodes a novel human IFN-α andits derivative, and specifically there can be mentioned DNA comprisingthe base sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or DNAencoding a protein comprising the amino acid sequence as set forth inSEQ ID NO: 3 or SEQ ID NO: 4.

[0034] Also encompassed in the scope of the present invention is DNAthat hybridizes to the DNA described above under a stringent condition,or DNA encoding a derivative of a human interferon-α selected from DNAencoding a protein in which one or a plurality of amino acid residues ofthe protein encoded by the above DNA have been replaced, deleted, and/oradded, wherein the protein encoded by said DNA has the characteristicsof (a) having a specific activity higher than 4.0×10⁸ units/mg asmeasured by an anti-viral activity assay on Sindbis virus using the FLcell, a human-derived cultured cell, and (b) migrating as a band with anapparent molecular weight of 20 kDa-23 kDa on a sodium dodecylsulfate-polyacrylamide gel electrophoresis after reduction treatment.The DNA of the present invention will now be sequentially explainedbelow.

[0035] 1) DNA Encoding the IFN-α Subtype IIIe

[0036] Among the above DNA, “DNA comprising the base sequence as setforth in SEQ ID NO: 1 or SEQ ID NO: 2” and “DNA encoding a proteincomprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQID NO: 4” are DNA encoding the human-derived IFN-α subtype IIIe of thepresent invention. Among them, the base sequence as set forth in SEQ IDNO: 1 and the amino acid sequence as set forth in SEQ ID NO: 3 are abase sequence and an amino acid sequence corresponding to thefull-length subtype IIIe including the signal peptide, and the basesequence as set forth in SEQ ID NO: 2 and the amino acid sequence as setforth in SEQ ID NO: 4 are a base sequence and an amino acid sequencecorresponding to the full-length mature type subtype IIIe including nosignal peptide.

[0037] Said DNA can be cloned by the PCR method described in Example 3below. As templates for performing PCR, genomic DNA or cDNA derived, forexample, from the Namalwa cell (ATCC No. CRL-1432 etc.) may be used, andas primers, a primer comprising the base sequence, for example, as setforth in SEQ ID NO: 6 and SEQ ID NO: 7 may be mentioned.

[0038] Furthermore, cloning may also be performed by modifying aminoacids based on the known IFN-α subtypes reported in Nature 290: 20-26(1981), etc. Said cloning may be readily performed by a person skilledin the art according to Molecular Cloning 2nd Ed., Cold Spring HarborLaboratory Press (1989) etc.

[0039] 2) DNA Encoding a Derivative of IFN-α Subtype IIIe

[0040] Among the above DNA, “DNA that hybridizes to the DNA of thesubtype IIIe under a stringent condition” and “DNA encoding a protein inwhich one or a plurality of amino acid residues of the amino acidsequence of the subtype IIIe have been replaced, deleted, and/or added”mean DNA encoding a protein having a structure similar to the subtypeIIIe such as an artificially constructed, so-called modified protein, anallele mutant present in the living body, and an IFN-α subtype similarto IIIe. Hereinbelow, protein having such a structure similar to thesubtype IIIe will be termed as a “derivative.”

[0041] As used herein, as a method of producing “DNA encoding protein inwhich one or a plurality of amino acid residues of the amino acidsequence of the subtype IIIe have been replaced, deleted, and/or added,”there can be known methods such as site-directed mutagenesis and the PCRmethod, which may be easily performed by a person skilled in the artaccording to Nucleic Acid Res. 10: 6487 (1982), Methods in Enzymology100: 448 (1983), Molecular Cloning 2nd Ed., Cold Spring HarborLaboratory Press (1989), PCR A Practical Approach, IRL Press, pp. 200(1991), etc.

[0042] As the number of amino acid residues to be modified, there can bementioned those numbers that may be replaced, deleted, and/or added byknown methods such as the site-directed mutagenesis mentioned above.Since IFN-α is a relatively small protein of which mature type comprises166 amino acids, the number of amino acid residues to be modified ispreferably 10 or less, and more preferably 5 or less. For sites that areimportant for activity expression, modification is preferablysubstitution to conservative amino acids.

[0043] As used herein, as a method of producing “DNA that hybridizes tothe DNA of the subtype IIIe under a stringent condition,” there can bementioned known methods such as a PCR method, and a hybridizationmethod. Specifically, it may be performed according to the methoddescribed in the above Molecular Cloning.

[0044] As used herein, “under a stringent condition” means a conditionin which hybridization is performed at 42° C. in a solution containing6×SSC (20×SSC represents 333 mM sodium citrate and 333 mM NaCl), 0.5%SDS, and 50% formamide, followed by washing at 68° C. in a solution of0.1×SSC and 0.5% SDS, a condition as described in the above-mentionedMolecular Cloning, or the like. More preferably, there can be mentioneda condition in which hybridization occurs only for those that aredifferent from the DNA of the subtype IIIe by about 1-5 amino acids.

[0045] Among the above DNA, the DNA encoding a protein which has thefollowing characteristics can be the DNA of the present invention: (a)having a specific activity higher than 4.0×10⁸ units/mg as measured byan anti-viral activity assay on Sindbis virus using human-derivedcultured cells, FL cells; and (b) migrating as a band with an apparentmolecular weight of 20 kDa-23 kDa on a sodium dodecylsulfate-polyacrylamide gel electrophoresis after reduction treatment.

[0046] Whether the protein encoded by the candidate DNA that can be theDNA of the present invention satisfies the above requirement (a) can beevaluated by performing an anti-viral activity assay as described below.

[0047] Thus, 45,000-60,000 FL cells (The National Institute of Health,ATCC etc.) prepared in a 10 v/v % bovine calf serum-Eagle's minimumessential medium are inoculated into each well of a microtiter plate,which is incubated in a 5% carbon dioxide incubator at 37° C. for 20hours. Then 100 μl of the candidate IFN sample is added to each well andincubated at 37° C. for 6 hours. The culture liquid is discarded and10⁵-10⁶ PFU of Sindbis virus (The National Institute of Health, ATTCetc.) per well is added, and incubated at 37° C. for 2 days. The cellsare stained in a 0.02 w/v % Neutral red-5 v/v % bovine calfserum-Eagle's minimum essential medium, and the degree of cytopathiceffect is determined by the amount of the dye incorporated.

[0048] As methods of calculating titer, the following method may bementioned. Thus, the dye incorporated into the cell is eluted with anacidified 30 v/v % ethanol and absorbance is determined at a wavelengthof 545 mμ. The experimental titer of the sample and the standard (TheNational Institute of Health) are calculated from the dilution factor ofthe sample exhibiting 50% of the absorbance of the dye incorporated intothe normal cell and that of the standard. The titer of the standards andthe experimental titer are used to determine a correction factor, whichis used to correct the experimental titers of the samples to obtain thetiters of the samples. In the above activity assay, those having aspecific activity higher than 4.0×10⁸ units/mg are included in the scopeof the present invention.

[0049] Whether the protein encoded by the candidate DNA satisfies theabove requirement (b) can be detected by subjecting the candidateprotein to reduction treatment with 2-mercaptoethanol followed by anormal sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and then staining with Coomassie brilliant blue. The densityof the SDS-PAGE gel at this time is preferably about 3.5% for theconcentration gel and about 15% for the separation gel. Electrophoresisis preferably performed at about 50 mA. In the SDS-PAGE as describedabove, those having an apparent molecular weight of 20 kDA-23 kDa areincluded in the scope of the present invention.

[0050] By recombinant DNA technology using the DNA of the presentinvention, the protein of the present invention, that is, the novelhuman IFN-α subtype IIIe and its derivative can be produced in largequantities. In order to produce the recombinant human IFN-α and itsderivative of the present invention by expressing the DNA of the presentinvention, methods are used, for example, based on many textbooks andreferences including the above Molecular Cloning. For human IFN-α2a and-α2b, their recombinant types have already been produced and arecommercially available. Based on the production method for known IFN-α,the novel human IFN-α and its derivative of the present invention can beproduced in large quantities (see Japanese Examined Patent Publication(Kokoku) No. 63-63198, Japanese Examined Patent Publication (Kokoku) No.3-21151, Nucleic Acids Res. 8: 4057 (1981), Nature 287: 411 (1980),Proc. Natl. Acad. Sci. USA 77: 5230 (1980), and the like).

[0051] Specifically, by optionally adding a regulatory gene such as apromoter sequence (for example, trp, lac, T7, and SV40 early promoter)that controls transcription to the upstream of the DNA to be expressed,which is then integrated into a suitable vector (for example, PBK-CMV,pCAGGS, and pZeoSV), it is possible to construct an expression vectorthat is replicated and expressed in the host cell. Then said expressionvector is introduced into a suitable host cell to obtain a transformant.As the host cell, there can be mentioned a prokaryote such asEscherichia coli, a unicellular eukaryote such as yeast, a multicellulareukaryote such as an insect or an animal, and the like. As the method ofintroducing an expression vector into a host cell, a known method can beused such as the calcium phosphate method, the DEAE-dextran method, andthe electric pulse method. By culturing the thus obtained transformantin a culture medium suitable for said transformant by a standard method,the desired recombinant human IFN-α and its derivative of the presentinvention can be produced. The recombinant human IFN-α and itsderivative of the present invention obtained in this manner can beisolated and purified by a common biochemical method using, for example,anti-IFN-α antibody.

[0052] Furthermore, they can also be obtained by purifying a subtypeobtained by using as raw material the human natural typelymphoblast-derived IFN-α (HLBI) (manufactured by SumitomoPharmaceutical Co., Ltd.) as described in Example 1 below.

[0053] The novel human IFN-α subtype IIIe and its derivative of thepresent invention thus obtained are encoded by the above DNA of thepresent invention, and they are proteins produced by the expression ofthe latter. As a specific example, there may be illustrated the novelhuman IFN-α subtype IIIe of the present invention comprising the aminoacid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0054] The novel IFN-α and its derivative of the present invention canbe used as active ingredient of pharmaceutical agents. Thus the presentinvention also intends to provide a pharmaceutical compositioncomprising a novel IFN-α and its derivative or a pharmaceuticallyacceptable salt thereof together with a pharmaceutically acceptablecarrier or excipient, and optionally with another therapeutic and/orpreventive agent.

[0055] It is conventionally known that IFN-α has a variety of effectsincluding an anti-viral effect, a cellular growth-suppressing effect, anatural killer cell-activating effect, and the like. Accordingly, thenovel IFN-α of the present invention is also expected to be able totreat various diseases based on these effects.

[0056] As the indicated diseases, cancer (malignant tumor), viraldiseases, and immunological diseases may be mentioned, and specificallythere can be mentioned kidney cancer, renal-cell carcinoma, breastcancer, bladder cancer, basal cell carcinoma, head and neck cancer,cervical dysplasia, skin carcinoma, Kaposi's sarcoma, malignantmelanoma, non-Hodgkin lymphoma, infant hemangioma, chronicgranulomatosis, type B chronic hepatitis, type C chronic hepatitis(active, non-active), herpes infections (genital herpes, corneal herpesinflammation, oral herpes inflammation, etc.), chronic myelocyticleukemia (CML), adult T cell leukemia, hairy cell leukemia, hairy cellleukemia, T cell leukemia virus (HTLV-1) myelopathy, multiple myeloma,lymphoma, subacute sclerosing panencephalitis (SSPE), Sjogren'ssyndrome, condyloma acuminata, AIDS, multiple sclerosis (MS),stomatitis, genital wart, intravaginal wart, erythrocytosis,thrombocythemia, psoriasis, mycosis fungoides, sudden deafness, seniledisciform macular degeneration, Paget's disease, and the like.

[0057] Since the IFN-α and its derivative of the present invention has aspecific activity higher than the conventional ones, they are expectedto be effective on cases with HCV-Genotype II, high virus level etc. onwhich conventional IFN is said to be not very effective.

[0058] The novel interferon-α and its derivative of the presentinvention or a pharmaceutically acceptable salt thereof may beadministered as a pharmaceutical composition via an oral or parenteral(for example, intravenous, subcutaneous or intramuscular injection,local, transrectal, transdermal, or nasal) route. As compositions fororal administration, there can be mentioned, for example, tablets,capsules, pills, granules, powders, liquids, and suspensions, and ascompositions for parenteral administration, there can be mentioned, forexample, aqueous or oily agents for injection, ointments, creams,lotions, aerosols, suppositories, and adhesives. It is also possible toprepare sustained release minipellet formulations and implant them nearthe affected area, or to gradually administer them to the affected areaon a continuous basis using an osmotic pump. These formulations may beprepared using conventionally known technology, and may containnon-toxic and inert carriers or excipients that are commonly used in thefield of pharmaceutics.

[0059] The above pharmaceutical compositions may be manufactured byblending the active ingredient of the present invention withpharmaceutically acceptable conventional carriers, excipients, binders,stabilizers, buffers, solution adjuvants, or tonicity agents. When usedas injections, there may be added buffers, solution adjuvants, tonicityagents etc..

[0060] The dosage and the frequency of administration may vary dependingon the condition and history of the disease, age and weight of thepatient, dosage form, etc., but when they are administered to adults(body weight 60 kg) via a parenteral (for example, intravenous) route,they are generally prepared, as appropriate, in the range of 0.001-1 mgper day, preferably 0.005-0.5 mg, and most preferably 0.010-0.2 mg, andadministered in single or several divided doses.

[0061] The present invention will now be explained in further detailshereinbelow with reference to examples, but it should noted that thepresent invention will not be limited by these examples in any way.

EXAMPLE 1 Isolation of Each IFN-α Subtype, and the Measurement of itsPhysical Properties and Biological Activity

[0062] In order to elucidate the physical properties and partialstructures in detail of major natural-type IFN-α subtypes contained inHLBI, IFN-α subtypes were isolated. It was found that each subtype canbe isolated by a reverse phase HPLC method using the μBondasphere (5μ,C4, 300A) column manufactured by Water's and the RP-304 column (Vydac-C4column) manufactured by Bio-Rad that have slightly differentsubtype-separation characteristics from each other, and therefore thepresent method was routinely used.

[0063] After extensive study, 12 IFN-α subtypes each being differentfrom one another were isolated and then each IFN-α subtype was subjectedto purity analysis using the reductive SDS-PAGE method. Furthermore,each IFN-α subtype was subjected to amino acid analysis, molecularweight determination by the reductive SDS-PAGE method, and the aminoacid sequencing of the amino terminal to clarify its physicalproperties. In addition, in order to elucidate the biological activity,the FL (amniotic) cell, a human-derived cultured cell, was used as atest cell and its anti-viral activity on Sindbis virus was determined.The experimental method and result are described below.

Experimental Method 1) IFN-α Samples

[0064] A total of two lots, a subpool lot No. PS/199 (50 ml) and asubpool lot No. PS/200 (50 ml) that are intermediate products of HLBIwere used as the samples for isolation of IFN-α subtypes.

2) Fractionation of IFN-α Sample (Lot No. PS/199)

[0065] After thawing, the sample was centrifuged at a high speed of12,000 rpm at 4° C. for 10 minutes, and 45 ml of the supernatantobtained was used. After passing 45 ml of the supernatant at a flow rateof 3 ml/min through the Water's μBondasphere (5μ, C4, 300A) column(19φ×150 mm) preequilibrated with a 0.1% TFA solution, 15 ml of the 0.1%TFA solution was run at a flow rate of 3 ml/min, and then the column wasextensively washed by passing 24 ml of the 0.1% TFA solution at a flowrate of 8 ml/min. Then by a gradient method in which acetonitrileconcentration was increased, IFN-α subtypes were eluted.

[0066] The eluate was monitored at A280 nm to fractionate at 4 ml/Fr.RP-HPLC was performed under the following condition.

RP-HPLC Condition

[0067] High performance liquid chromatography instrument: LC6Amanufactured by Shimadzu

[0068] Column: Water's μBondasphere (5μ, C4, 300A) column (19φ×150 mm)

[0069] Flow rate: 8 ml/min

[0070] Eluent A: 0.1% TFA, eluent B: 0.1% TFA-95% acetonitrile

[0071] Gradient elution condition: As described in Table 1 TABLE 1 Time:min 0 3 35 75 95 105 115 115.1 Solution 0 30 46 52 55 60 100 0 B %

[0072] Detection: A280 nm, 0.04 aufs

[0073] Fractionation: 4 ml/Fr

[0074] 3) Fractionation of the IFN-α Sample (Lot No. PS/200)

[0075] After thawing, the sample was centrifuged at a high speed of12,000 rpm at 4° C. for 10 minutes, and 45 ml of the supernatantobtained was used. After passing 45 ml of the supernatant at a flow rateof 3 ml/min through the Water's μBondasphere (5μ, C4, 300A) column(19φ×150 mm) preequilibrated with a 0.1% TFA solution, 15 ml of the 0.1%TFA solution was run at a flow rate of 3 ml/min, and then the column wasextensively washed by passing 24 ml of the 0.1% TFA solution at a flowrate of 8 ml/min. Then by a gradient method in which acetonitrileconcentration was increased, IFN-α subtypes were eluted.

[0076] The eluate was monitored at A280 nm to fractionate at 4 ml/Fr.RP-HPLC was performed under the above condition 2).

4) Isolation of IFN-α Subtypes

[0077] Fractions obtained in the above 2) and 3) were subjected topurification by the reverse phase HPLC method using the RP-304 columnmanufactured by Bio-Rad. An equal amount of a 0.1% TFA solution wasadded to each fraction, and then equilibrated with the 0.1% TFAsolution. After running at a flow rate of 1 ml/min through the Bio-Rad'sRP-304 (5μ, C4, 300A) column (4.6φ×250 mm), the column was extensivelywashed by passing 15 ml of the 0.1% TFA solution at a flow rate of 1ml/min. Then by a gradient method in which acetonitrile concentrationwas increased, IFN-α subtypes were eluted. The eluate was monitored atA220 nm to fractionate at 0.3 or 0.4 ml/Fr. RP-HPLC was performed foreach fraction under the following condition. Furthermore, each IFN-αsubtype was chromatographed until a single peak was obtained on RP-HPLC.

RP-HPLC Condition

[0078] High performance liquid chromatography instrument: 5000 LCmanufactured by Varian

[0079] Column: RP-304 (5μ, C4, 300A) column (4.6φ×250 mm) manufacturedby Bio-Rad

[0080] Flow rate: 1 ml/min

[0081] Eluent A: 0.1% TFA, eluent B: 0.1% TFA-95% acetonitrile

[0082] Gradient elution condition: As described in Table 2 TABLE 2 Time:min 0 3 5 45 65 Solution 0 40 46 50 52 B %

[0083] Detection: A220 nm, 0.64-2.56 aufs

[0084] Fractionation: 0.3 or 0.4 ml/Fr

5) Purity Analysis by the Reverse Phase HPLC of IFN-α Subtypes

[0085] The above isolated IFN-α subtypes were subjected to purityanalysis by a reverse phase HPLC method using the Bio-Rad's RP-304 (5μ,C4, 300A) column.

RP-HPLC Condition

[0086] High performance liquid chromatography instrument: LC4Amanufactured by Shimadzu

[0087] Column: RP-304 manufactured by Bio-Rad (5μ, C4, 300A) column(4.6φ×250 mm)

[0088] Flow rate: 1 ml/min

[0089] Eluent A: 0.1% TFA-40% acetonitrile, eluent B: 0.1% TFA-50%acetonitrile

[0090] Gradient elution condition: As described in Table 3 TABLE 3 Time:min 0 2 42 42.1 Solution 0 20 100 0 B %

[0091] Detection: A220 nm

6) Purity Analysis by the SDS-PAGE Method of the IFN-α Subtype

[0092] The above isolated IFN-α subtypes were subjected to purityanalysis by the SDS-PAGE method. After about 0.1-0.3 μg of each IFN-αsubtype was concentrated without heating by a speedvac concentratorunder reduced pressure, 2-mercaptoethanol was added and then reduced at100° C. for 1.5 minutes. An electrophoresis gel having a concentratinggel concentration of 3.5% and a separating gel concentration of 15% wasused. After preelectrophoresis at 40 mA, SDS-PAGE analysis was performedby electrophoresing at 50 mA. After the electrophoresis, it was stainedwith a CBB solution.

7) Analysis of the Amino Acid Composition of IFN-α Subtypes

[0093] To 20-100 μl of the IFN-α subtype solutions isolated above, 1nmol of Nle was added as an internal standard, and concentrated by aspeedvac concentrator under reduced pressure. 0.2 ml of constant boilinghydrochloric acid containing 0.1% thioglycolic acid was added, and thensealed under reduced pressure and hydrolyzed at 110° C. for 24 hours.Using the automatic amino acid analyzer model 835 manufactured byHitachi Seisakusho, the constituent amino acids that formed wereanalyzed by the OPA hypo method, and amino sugars and Trp were analyzedby the OPA method. Based on the analytical result of amino acidcomposition, the protein concentration of each IFN-α subtype wascalculated.

8) Amino Terminal-Amino Acid Sequencing of IFN-α Subtypes

[0094] The above isolated IFN-α subtypes were subjected to automaticEdman degradation using the gas-phase type protein sequencer model 477Amanufactured by Applied Biosystems, and the PTH-amino acids that formedwere identified using the PTH-amino acid analyzer model 120Amanufactured by Applied Biosystems.

9) Anti-Viral Activity of IFN-α Subtypes

[0095] As the test cell for anti-viral activity, the FL (amniotic) cell,a human-derived cultured cell, was used. The cell was provided by theNational Institute of Health. The medium used was a MEM mediumcontaining 10% fetal calf serum. Cell culture was performed at 37° C.under 5% CO2. The virus used was Sindbis virus (SBV) provided from theNational Institute of Health. SBV was used to prepared a virus stockusing developing chicken eggs. As the standard IFN for anti-viralactivity determination, the national standard Lot. J-501 (obtained fromthe National Institute of Health) was used. Specific activity wasexpressed per mg of protein with the concentration that inhibits 50% ofthe virus being defined as one unit. The detailed method of measurementis shown below.

[0096] First, 45,000-60,000 FL cells prepared in a 10 v/v % bovine calfserum-Eagle's minimum essential medium were inoculated into each well ofa microtiter plate, which was incubated in a 5% carbon dioxide incubatorat 37° C. for 20 hours. Then 100 μl of each IFN sample was added to eachwell and incubated at 37° C. for 6 hours. The culture liquid wasdiscarded and 10⁵-10⁶ PFU of Sindbis virus per well was added, andincubated at 37° C. for 2 days. The cells were stained in a 0.02 w/v %Neutral red −5 v/v % bovine calf serum-Eagle's minimum essential medium,and the degree of cytopathic effect was determined by the amount of thedye incorporated.

[0097] Titer was calculated as follows. Thus, the dye incorporated intothe cell was eluted with an acidified 30 v/v % ethanol and absorbancewas determined at a wavelength of 545 mμ. The experimental titer of thesample and the standard were calculated from the dilution factor of thesample exhibiting 50% of the absorbance of the dye incorporated into thenormal cell and that of the standard. The titer of the standard and itsexperimental titer were used to determine a correction factor, which wasused to correct the experimental titers of the sample to obtain thetiters of the samples.

Experimental Results and Discussion 1) Isolation of IFN-α Subtypes

[0098] According to the above experimental methods 2) and 3), thesupernatant of the subpool lot No. PS/199 (45 ml) and the supernatant ofthe subpool lot No. PS/200 (45 ml) were fractionated by the Water'sμBondasphere (5μ, C4, 300A) column (19φ×150 mm). There were ten majorfractions: fr.4, fr.8, fr.9, fr.11, fr.14, fr.18, fr.19, fr.21, fr.24,and fr.25.

[0099] Then, according to the above experimental method 4), RP-HPLCRP-HPLC preparation was performed for each fraction using the Bio-Rad'sRP-304 (5μ, C4, 300A) column to obtain 12 IFN-α subtype fractions. As aresult of purity analysis performed in accordance with the method in 5)above for each fraction by the reverse phase HPLC method using theRP-304 (5μ, C4, 300A) column, every sample exhibited a single peakconfirming a high purity.

[0100] Furthermore, according to the above experimental method 6),purity analysis by the SDS-PAGE method was performed for each fractionafter reduction treatment, and every sample exhibited a single bandconfirming the high purity. The molecular weight obtained by theSDS-PAGE method is shown in Table 4 below. TABLE 4 IFN-α subtypeMolecular weight fr. No. kDa  4 26.8  8A 22.3  9 21.6 11A 22.6 11B 20.914 22.4 18A 21.2 18′B 22.2 19B1 20.9 21 28.2 24 23.0 25 22.6

2) Amino Terminal-Amino Acid Sequence Analysis of IFN-α Subtypes

[0101] For 12 IFN-α subtypes isolated, the amino acid sequence of theamino terminal was analyzed according to the above experimental method8) to obtain the following result.

[0102] The amino terminal-amino acid sequence of IFN-α subtypes fr.8Aand fr.9 coincided with the previously published sequence of IFN-α2b.

[0103] The amino terminal-amino acid sequence of IFN-α subtype fr.4coincided with the previously published sequence of IFN-α4.

[0104] The amino terminal-amino acid sequence of IFN-α subtype fr.11Acoincided with the previously published sequence of IFN-α21.

[0105] The amino terminal-amino acid sequence of IFN-α subtype fr.11Bcoincided with the previously published sequence of IFN-α5.

[0106] The amino terminal-amino acid sequence of IFN-α subtype fr.18Acoincided with the previously published sequence of IFN-α17.

[0107] The amino terminal-amino acid sequence of IFN-α subtype fr.18′Bcoincided with the previously published sequence of IFN-α7.

[0108] The amino terminal-amino acid sequence of IFN-α subtype fr.19B1coincided with the previously published sequence of IFN-α17.

[0109] The amino terminal-amino acid sequence of IFN-α subtype fr.21coincided with the previously published sequence of IFN-α8.

[0110] The amino terminal-amino acid sequence of IFN-α subtypes fr.24and fr.25 coincided with the previously published sequence of IFN-α1.

[0111] The amino terminal-amino acid sequence of IFN-α subtype fr.14 wassimilar to that of the previously published IFN-α10a (=−αC) (Nature 1981Mar. 5: 290, 20-26), except that the sequence was novel in that theamino acid at position 19 was Ala in stead of Gly. We have termed thisnovel subtype IIIe. The amino terminal-amino acid sequence of thesubtype IIIe is shown in SEQ ID NO: 5.

3) Anti-Viral Activity of IFN-α Subtypes

[0112] According to the above experimental method 9), anti-viralactivity on Sindbis virus (SBV) was determined. The anti-viral activityof each IFN subtype is shown in Table 5 below.

[0113] All IFN subtypes showed anti-viral activity and the anti-viralactivity of fr.8A (IFN-α2b), a major IFN subtype of HLBI, was 1.67×10⁸u/mg. The IFN-α subtype that had the highest specific activity was fr.14(IIIe), and its anti-viral activity was 5.21×10⁸ u/mg which is higherthan that of any other conventionally known IFN-α subtypes. On the otherhand, the IFN-α subtype that had the lowest specific activity was fr.24,and its anti-viral activity was 0.12×10⁸ u/mg. Thus, it was demonstratedthat the novel IFN subtype fr.14 (IIIe) had the highest anti-viralactivity and IFN subtype fr.24 whose amino terminal-amino acid sequencecoincides with that of IFN-α1 had the lowest anti-viral activity. TABLE5 Classification based on the amino IFN-α subtype terminal-amino acidActivity × fr. No sequence 10⁸ u/mg  4 IFN-α14 1.73  8A IFN-α2b 1.67  9IFN-α2b 1.83 11A IFN-α21 1.69 11B IFN-α5 1.44 14 (IIIe) 5.21 18A IFN-α172.63 18′B IFN-α7 1.65 19B1 IFN-α17 2.57 21 IFN-α8 1.50 24 IFN-α1 0.12 25IFN-α1 0.20

EXAMPLE 2 Analysis of the Primary Structure of the IFN-α Subtype IIIe

[0114] The IFN-α subtype IIIe obtained in Example 1 is a subtype thathas a novel amino acid sequence and exhibits an unprecedentedly highspecific activity. In order to determine the primary structure of saidIIIe, the following experiment was performed.

Experimental Method 1) Analysis of Primary Structure of the IFN-αSubtype IIIe

[0115] As the method of analyzing primary structure, a method ofcleaving the Met residue with cyanogen bromide and a method of cleavingthe basic amino acid residue with trypsin were employed.

The Method of Cleaving a Met Residue with Cyanogen Bromide

[0116] 30 μg of the IFN-α subtype IIIe was dissolved in 200 μl of 70%formic acid, to which 1 μmole of cyanogen bromide was added and allowedto stand at 24° C. for 20 hours. After the disappearance of the rawmaterial IIIe was confirmed by a reverse phase HPLC using the PR-304column, 9 volumes of water was added to stop the reaction and thenconcentrated under reduced pressure by a speedvac concentrator equippedwith a NaOH trap.

[0117] To the cyanogen bromide-fragmented peptides, 0.2 ml of a 0.5 MTris solution (pH 8.1) containing argon gas-displaced 6M guanidine and 2mM EDTA was added and dissolved. After adding 0.4 μmole of DTT anddisplaced with argon gas, it was placed in the dark and was reduced at37° C. for 3 hours. 0.8 μmole of monoiodo acetamide that wasrecrystalized was added, and alkylated in the dark at 37° C. for 1 hour.After passing the reaction solution at a flow rate of 1 ml/min throughthe RP-304 (5μ, C4, 300A) column (4.6 φ×250 mm) manufactured by Bio-Radpreviously equilibrated with a 0.1% TFA solution, the column wasextensively washed by passing 15 ml of the 0.1% TFA solution at a flowrate of 1 ml/min. Then, by a gradient method in which acetonitrileconcentration was increased, the constituent peptides were eluted. Theeluate was monitored at A220 nm. It was purified under the followingRP-HPLC condition.

RP-HPLC Condition

[0118] High performance liquid chromatography instrument: 5000 LCmanufactured by Varian

[0119] Column: RP-304 (5μ, C4, 300A) column (4.6 φ×250 mm) manufacturedby Bio-Rad

[0120] Flow rate: 1 ml/min

[0121] Eluent A: 0.1% TFA, eluent B: 0.1% TFA-95% acetonitrile

[0122] Gradient elution condition: As described in Table 6 TABLE 6 Time:min 0 60 70 Solution B % 0 60 100

[0123] Detection: A220 nm, 1.28 aufs and A280 nm, 0.16 aufs

2) The Method of Cleaving with Trypsin and the ReductiveCarboxymethylation Method

[0124] 60 μg of the subtype IIIe was dissolved in 200 μl of a 0.2 MNaHCO₃ (pH 8.3) solution, to which 2 μg Of TPCK-Trypsin was added andthe mixture was allowed to stand at 37° C. for 24 hours. To thetrypsin-fragmented peptides, 0.05 ml of a 0.5 M Tris solution (pH 8.1)containing argon gas-displaced 6M guanidine and 2 mM EDTA was added anddissolved. After adding DTT at an amount of 50 times that of the Cysresidue and displaced with argon gas, it was placed in the dark and wasreduced at 37° C. for 1 hour. Monoiodo acetamide that was recrystalizedwas added at an amount of 100 times that of the Cys residue, andalkylated in the dark at 37° C. for 30 minutes. After passing thereaction solution at a flow rate of 1 ml/min through the RP-304 (5μ, C4,300A) column (4.6 φ×250 mm) manufactured by Bio-Rad previouslyequilibrated with a 0.1% TFA solution, the column was extensively washedby passing 15 ml of the 0.1% TFA solution at a flow rate of 1 ml/min.Then, by a gradient method in which acetonitrile concentration wasincreased, the constituent peptides were eluted. The eluate wasmonitored at A220 nm. It was purified under the following RP-HPLCcondition.

RP-HPLC Condition

[0125] High performance liquid chromatography instrument: 5000 LCmanufactured by Varian

[0126] Column: RP-304 (5μ, C4, 300A) column (4.6 ®×250 mm) manufacturedby Bio-Rad

[0127] Flow rate: 1 ml/min

[0128] Eluent A: 0.1% TFA, eluent B: 0.1% TFA-95% acetonitrile

[0129] Gradient elution condition: As described in Table 7 TABLE 7 Time:min 0 10 40 60 Solution 0 25 50 100 B %

[0130] Detection: A220 nm, 0.08-0.16 aufs

3) Analysis of Amino Acid Composition

[0131] To 20-100 μl of the fragmented peptide solution of the purifiedIFN-α subtype IIIe, 1 nmol of Nle was added as an internal standard,which was then concentrated under reduced pressure in a speedvacconcentrator. 0.2 ml of constant boiling hydrochloric acid containing0.1% thioglycolic acid was added, and then sealed and hydrolyzed at 110°C. for 24 hours. Using the automatic amino acid analyzer model 835manufactured by Hitachi Seisakusho, the constituent amino acids thatformed were analyzed by the OPA hypo method, and amino sugars and Trpwere analyzed by the OPA method.

4) Sequencing of Amino Terminal-Amino Acids

[0132] The fragmented peptide solution of the purified IFN-α subtypeIIIe were subjected to automatic Edman degradation using the gas-phasetype protein sequencer model 477A manufactured by Applied Biosystems,and the PTH-amino acids that formed were identified using the PTH-aminoacid analyzer model 120A manufactured by Applied Biosystems.

Experimental Results and Discussion 1) Structural Analysis of CyanogenBromide-Fragmented Peptides

[0133] After 30 μg of the IFN-α subtype IIIe was fragmented withcyanogen bromide, the reductive-carboxymethylated peptides were purifiedwith the RP-304 (5μ, C4, 300A) column (4.6 φ×250 mm) manufactured byBio-Rad. Each peptide was subjected to amino acid analysis, amino sugaranalysis, and amino acid sequencing. For peak fractions corresponding toSEQ ID NO: 21-60, SEQ ID NO: 61-106, SEQ ID NO: 113-149, and SEQ ID NO:150-166, amino acid sequences were identified.

2) Structural Analysis of Trypsin-Fragmented Peptides

[0134] After 80 μg of the IFN-α subtype IIIe was fragmented withtrypsin, the reductive-carboxymethylated peptide was purified with theRP-304 column. Each peptide was subjected to amino acid analysis, aminosugar analysis, and amino acid sequencing. For peak fractionscorresponding to SEQ ID NO: 1-12, SEQ ID NO: 14-23, SEQ ID NO: 24-50,SEQ ID NO: 51-84, SEQ ID NO: 85-121, SEQ ID NO: 122-126, SEQ ID NO:136-145, SEQ ID NO: 146-150 and SEQ ID NO: 151-160, amino acid sequenceswere identified.

[0135] As hereinabove described, the cyanogen bromide-fragmentation andtrypsin-fragmentation of the IFN-α subtype IIIe and the followingstructural analysis of the constituent peptides confirmed that the IFN-αsubtype IIIe is comprised of 166 amino acid residues. The identificationresult of all amino acid sequences is shown in SEQ ID NO: 4. The primarystructure of the subtype IIIe was similar to that of the previouslypublished IFN-α10a (=−αC) (Nature 1981 Mar. 5: 290, 20-26), except thatthe sequence was novel in that the amino acid at position 19 was Ala instead of Gly.

EXAMPLE 3 Gene Cloning of the IFN-α Subtype IIIe

[0136] The gene of the IFN-α subtype IIIe was specifically amplified byPCR. As primers, U-10 (SEQ ID NO: 6) and L-10 (SEQ ID NO: 7), sequencesthat are specific to the subtype IIIe, were used. Since the IFN-α genecontained no introns, genomic DNA was used as a template for PCR. Theexperimental method is described below.

[0137] The genomic DNA of Namalwa cell was prepared using the DNAExtraction kit (Stratagene) and was used as the template for PCR. PCRwas performed under the following condition using KOD DNA polymerase(Toyobo), and primers U-10 and L-10. Thus, a reaction mixture comprising1 μg of the template DNA, 0.5 μg of each primer, 1×KOD buffer, 1 mMMgCl₂, 200 μM of each dNTP, and 2.5 U KOD was subjected to, afterheating at 90° C. for 3 minutes, 30 cycles of PCR with each cyclecomprising 95° C. for 30 seconds, 68° C. for 30 seconds, and 72° C. for90 seconds, and then cooled at 4° C.

[0138] This PCR product was cloned into pUC18 vector to obtain arecombinant plasmid of the IFN-α subtype IIIe.

[0139] Thereafter, using the ABI PRISM Dye Terminator Cycle SequencingReady Reaction Kit (Perkin-Elmer) the above plasmid was subjected to adye-terminator reaction, and using the ABI PRISM 377 DNA Sequencer(Perkin-Elmer) the base sequence was analyzed. The determined basesequence (567 bp) is shown in SEQ ID NO: 1, and the amino acid sequence(189 amino acids) deduced from said base sequence is shown in SEQ ID NO:3. The sequence of the amino acid sequence at position 24 and after ofSEQ ID NO: 3 completely coincided with the amino acid sequence (SEQ IDNO: 4) determined in the above Example 2. The sequence at positions 1-23of the amino acid sequence as set forth in SEQ ID NO: 3 corresponds tothe signal sequence.

[0140] In the comparison of the base sequence of IIIe as set forth inSEQ ID NO: 1 with the previously reported base sequence of IFN-α10a,three bases (positions 66, 96, and 125) were different.

[0141] As described above, for the novel IFN-α subtype IIIe, a completeDNA sequence containing the signal sequence portion was obtained.

Industrial Applicability

[0142] The present invention can provide a novel human IFN-α and itsderivative having an unprecedentedly high specific activity, and apharmaceutical composition comprising said IFN-α and its derivative asactive ingredient.

[0143] Sequence Listing Free Text

[0144] The amino acids at positions 1, 29, and 37 as set forth in SEQ IDNO: 5 are unknown.

1 7 1 567 DNA Homo sapiens 1 atg gcc ctg tcc ttt tct tta ctt atg gcc gtgctg gtg ctc agc tac 48 Met Ala Leu Ser Phe Ser Leu Leu Met Ala Val LeuVal Leu Ser Tyr 5 10 15 aaa tcc atc tgt tct cta ggc tgt gat ctg cct cagacc cac agc ctg 96 Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gln ThrHis Ser Leu 20 25 30 ggt aat agg agg gcc ttg ata ctc ctg gca caa atg ggaaga atc tct 144 Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gln Met Gly ArgIle Ser 35 40 45 cct ttc tcc tgc ctg aag gac aga cat gat ttc cga atc ccccag gag 192 Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Arg Ile Pro GlnGlu 50 55 60 gag ttt gat ggc aac cag ttc cag aag gct caa gcc atc tct gtcctc 240 Glu Phe Asp Gly Asn Gln Phe Gln Lys Ala Gln Ala Ile Ser Val Leu65 70 75 80 cat gag atg atc cag cag acc ttc aat ctc ttc agc aca gag gactca 288 His Glu Met Ile Gln Gln Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser85 90 95 tct gct gct tgg gaa cag agc ctc cta gaa aaa ttt tcc act gaa ctt336 Ser Ala Ala Trp Glu Gln Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu 100105 110 tac cag caa ctg aat gac ctg gaa gca tgt gtg ata cag gag gtt ggg384 Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Glu Val Gly 115120 125 gtg gaa gag act ccc ctg atg aat gag gac tcc atc ctg gct gtg agg432 Val Glu Glu Thr Pro Leu Met Asn Glu Asp Ser Ile Leu Ala Val Arg 130135 140 aaa tac ttc caa aga atc act ctt tat cta ata gag agg aaa tac agc480 Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Ile Glu Arg Lys Tyr Ser 145150 155 160 cct tgt gcc tgg gag gtt gtc aga gca gaa atc atg aga tcc ctctcg 528 Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser165 170 175 ttt tca aca aac ttg caa aaa aga tta agg agg aag gat 567 PheSer Thr Asn Leu Gln Lys Arg Leu Arg Arg Lys Asp 180 185 2 498 DNA Homosapiens 2 tgt gat ctg cct cag acc cac agc ctg ggt aat agg agg gcc ttgata 48 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 510 15 ctc ctg gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag gac 96Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30aga cat gat ttc cga atc ccc cag gag gag ttt gat ggc aac cag ttc 144 ArgHis Asp Phe Arg Ile Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 cagaag gct caa gcc atc tct gtc ctc cat gag atg atc cag cag acc 192 Gln LysAla Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 ttc aatctc ttc agc aca gag gac tca tct gct gct tgg gaa cag agc 240 Phe Asn LeuPhe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser 65 70 75 80 ctc ctagaa aaa ttt tcc act gaa ctt tac cag caa ctg aat gac ctg 288 Leu Leu GluLys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 gaa gca tgtgtg ata cag gag gtt ggg gtg gaa gag act ccc ctg atg 336 Glu Ala Cys ValIle Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 aat gag gactcc atc ctg gct gtg agg aaa tac ttc caa aga atc act 384 Asn Glu Asp SerIle Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctt tat ctaata gag agg aaa tac agc cct tgt gcc tgg gag gtt gtc 432 Leu Tyr Leu IleGlu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 aga gca gaaatc atg aga tcc ctc tcg ttt tca aca aac ttg caa aaa 480 Arg Ala Glu IleMet Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 aga ttaagg agg aag gat 498 Arg Leu Arg Arg Lys Asp 165 3 189 PRT Homo sapiens 3Met Ala Leu Ser Phe Ser Leu Leu Met Ala Val Leu Val Leu Ser Tyr 5 10 15Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gln Thr His Ser Leu 20 25 30Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gln Met Gly Arg Ile Ser 35 40 45Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Arg Ile Pro Gln Glu 50 55 60Glu Phe Asp Gly Asn Gln Phe Gln Lys Ala Gln Ala Ile Ser Val Leu 65 70 7580 His Glu Met Ile Gln Gln Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser 85 9095 Ser Ala Ala Trp Glu Gln Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu 100105 110 Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Glu Val Gly115 120 125 Val Glu Glu Thr Pro Leu Met Asn Glu Asp Ser Ile Leu Ala ValArg 130 135 140 Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Ile Glu Arg LysTyr Ser 145 150 155 160 Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile MetArg Ser Leu Ser 165 170 175 Phe Ser Thr Asn Leu Gln Lys Arg Leu Arg ArgLys Asp 180 185 4 166 PRT Homo sapiens 4 Cys Asp Leu Pro Gln Thr His SerLeu Gly Asn Arg Arg Ala Leu Ile 5 10 15 Leu Leu Ala Gln Met Gly Arg IleSer Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Ile Pro GlnGlu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Gln Ala Ile Ser ValLeu His Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Glu AspSer Ser Ala Ala Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu Lys Phe Ser ThrGlu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln GluVal Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile LeuAla Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Ile GluArg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu IleMet Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg LeuArg Arg Lys Asp 165 5 40 PRT Homo sapiens MOD_RES (1) Unknown amino acid5 Xaa Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 5 1015 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Xaa Leu Lys Asp 20 2530 Arg His Asp Phe Xaa Ile Pro Gln 35 40 6 30 DNA Homo sapiens 6ataggatcca ggccgaagtt caaggttatc 30 7 34 DNA Homo sapiens 7 tacaagcttcaggatcattg ccatgttgaa ccag 34

1. A purified polypeptide comprising an amino acid sequence as set forthin SEQ ID NO: 3 or SEQ ID NO:
 4. 2. A purified polypeptide comprising anamino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4,wherein from one to five amino acid residues have been replaced,deleted, and/or added, and wherein the polypeptide has the followingcharacteristics (a) and (b): (a) a specific activity higher than 4.0×108units/mg in an anti-Sindbis virus assay on cultured FL cells; and (b) anapparent molecular weight of 20 kDa-23 kDa as determined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) aftertreatment with a reducing agent.
 3. A pharmaceutical compositioncomprising the polypeptide of claim 1 or claim 2, and a pharmaceuticallyacceptable carrier or excipient.
 4. The pharmaceutical composition ofclaim 3, which is for treatment of a viral disease.
 5. Thepharmaceutical composition of claim 3, which is for treatment of cancer.6. A method for treating a viral disease in a mammal comprisingadministering to said mammal in need of treatment an effective amount ofthe polypeptide of claim 1 or
 2. 7. The method of claim 6, wherein saidmammal is a human.
 8. A method for treating cancer in a mammalcomprising administering to said mammal in need of treatment aneffective amount of the polypeptide of claim 1 or claim
 2. 9. The methodof claim 8, wherein said mammal is a human.
 10. A recombinantpolypeptide produced by a process comprising: (a) transforming a cellwith an expression construct, said expression construct comprising anucleotide sequence operably linked to a promoter, said nucleotidesequence encoding the polypeptide with an amino acid sequence as setforth in SEQ ID NO: 3 or SEQ ID NO: 4, thereby creating a transformant;(b) culturing said transformant; and (c) recovering expressedpolypeptide.
 11. A recombinant polypeptide produced by a processcomprising: (a) transforming a cell with an expression construct, saidexpression construct comprising a nucleotide sequence operably linked toa promoter; said nucleotide sequence encoding the polypeptide with anamino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4,wherein from one to five amino acid residues have been replaced,deleted, and/or added, and wherein the polypeptide has the followingcharacteristics (1) and (2): (1) a specific activity higher than 4.0×108units/mg in an anti-Sindbis virus assay on cultured FL cells; and (2) anapparent molecular weight of 20 kDa-23 kDa as determined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) aftertreatment with a reducing agent; thereby creating a transformant; (b)culturing said transformant; and (c) recovering expressed polypeptide.12. A pharmaceutical composition comprising the recombinant polypeptideof claim 10 or claim 11, and a pharmaceutically acceptable carrier orexcipient.
 13. The pharmaceutical composition of claim 12, which is fortreatment of a viral disease.
 14. The pharmaceutical composition ofclaim 12, which is for treatment of cancer.
 15. A method for treating aviral disease in a mammal comprising administering to said mammal inneed of treatment an effective amount of the recombinant polypeptide ofclaim 10 or claim
 11. 16. The method of claim 15, wherein said mammal isa human.
 17. A method for treating cancer in a mammal comprising;administering to said mammal in need of treatment an effective amount ofthe recombinant polypeptide of claim 10 or claim
 11. 18. The method ofclaim 17, wherein said mammal is a human.